Innate immunity is the first-line of defense used by all types of cells to protect against invading microbes. Defense against microbial attack is critically important for the entire body and touches far-ranging medical domains. Properly regulated antimicrobial responses control infection and limit the effects of injury and irritation. When these processes go awry - because the response is too weak, too strong or misplaced - the result may be immunodeficiency, autoimmunity, or cancer. Studies of innate antiviral immune responses have been highly influenced by the discovery of the IKK-related kinase, TBK1. Substrates for TBK1 include the transcription factor interferon regulatory factor (IRF)-3, which controls transcription of type 1 interferons (IFN). This proposal investigates the molecular mechanisms underlying TBK1-dependent transcription of IFN and other antimicrobial cytokines. Our preliminary data identify two E3 ubiquitin ligases which target TBK1 for K63-linked polyubiquitination (pUb), control TBK1 kinase activity, regulate IRF3 signaling, and protect against viral infection. Specific Aim 1 analyzes TBK1 pUb including characterization of the E3 ligases and identification of TBK1 Ub acceptor sites. As background we provide evidence that TBK1 is posttranslationally modified by K63-linked pUb chains in a ligand dependent fashion. We also supply data indicating a role for mind bomb (MIB) proteins in regulating TBK1 pUb and IFN production. This is the first evidence that the E3 ligases MIB1 and MIB2 participate in IRF dependent responses. Specific Aim 2 investigates the consequences of MIB1 and/or MIB2 deficiency on IFN production and protection against viral replication. Initial experiments have reassuringly supported a role for mib genes in protection against vesicular stomatitis virus (VSV). Specific Aim 3 will examine the requirements for MIB-mediated TBK1 pUb in response to RLR and TLR ligands or in response to dsDNA in fibroblasts, macrophages, and dendritic cells. The final Specific Aim will identify TBK1 associated molecules which regulate the RLR, TLR3, TLR4, or dsDNA signaling pathways. The molecular mechanisms controlling the assembly of TBK1 signalosomes will be determined. Our recent data suggest a novel hypothesis accounting for MAVS-dependent TBK1 activation; the proposed studies will test and expand upon this model. Detailed descriptions of the molecular interactions that regulate TBK1 activity are important since dysregulation of this kinase is associated with susceptibility to viral infection and other diseases. Insights from the proposed studies may ultimately facilitate design of therapies that may curtail IKK-related pathways in chronic inflammatory diseases or enhance TBK1 activity when needed to boost immunity.